【作者】 吴剑平；

【导师】 杨斐；

【作者基本信息】 复旦大学 ， 动物学， 2011， 硕士

【摘要】 目的：环境因素与实验动物的福利水平密切相关。常规实验操作和饲育环境是可能影响动物福利的环境因素中最重要的两种因素。评价这些环境因素对动物的影响将有助于提高实验动物的福利水平。但目前有关这方面的系统研究均很少见。本研究即通过观察不同的常规实验操作(腹腔注射、肌肉注射、灌胃、剪尾采血、尾静脉切割采血、硬固定和软固定)和饲育环境(屏障环境中采用普通开放饲养笼饲育、屏障环境中采用独立通风笼具(IVC)饲育、普通环境中采用IVC饲育)对Wistar大鼠生理稳态的影响,初步分析这些环境因素对Wistar大鼠福利的影响及对相关实验研究可能会造成的背景性干扰。方法：1.将Wistar大鼠按给药操作类别进行分组,分别对各组动物进行腹腔注射、肌肉注射、灌胃3种操作,连续处理7天,测定这些操作对Wistar大鼠神经内分泌指标[皮质酮(CORT)、β-内啡肽(β-EP)]、免疫指标[白介素-2(IL-2)、白介素-4(IL-4)、γ-干扰素(IFN-γ)、γ-干扰素/白介素-4(IFN-γ/IL-4)]、白细胞相关指标[白细胞总数(WBC)、中性粒细胞比例(WLCR)、淋巴细胞比例(WSCR)]、红细胞相关指标[红细胞总数(RBC)、血红蛋白含量(HGB)、红细胞压积(HCT)]和应激蛋白[C-反应蛋白(CRP)]与应激分子(hsp72mRNA表达量)的影响,并评价动物福利状态。2.将Wistar大鼠按采血操作类别进行分组,分别对各组动物进行剪尾采血和尾静脉切割采血,连续处理7天,同样测定这些操作对Wistar大鼠神经内分泌指标、免疫指标、白细胞相关指标、红细胞相关指标、应激蛋白和应激分子的影响,评价动物福利状态。3.将Wistar大鼠按固定操作类别进行分组,分别对各组动物进行硬固定和软固定操作,连续处理7天,同样测定这些操作对Wistar大鼠神经内分泌指标、免疫指标、白细胞相关指标、红细胞相关指标、应激蛋白和应激分子的影响,评价动物福利状态。4.将Wistar大鼠按不同的饲育环境进行分组,动物分别饲养在屏障环境设施内的普通开放饲养笼中、屏障环境设施内的IVC中和普通环境设施内的IVC中,各组均饲养45天,之后测定各组Wistar大鼠神经内分泌指标(CORT、β-EP)、免疫指标[白介素-1α(IL-1α)、白介素-1β(IL-1β)、IL-2、IL-4、白介素-10(IL-10)、白介素-13(IL-13)、IFN-γ、IFN-γ/IL-4、单核细胞趋化因子-1(MCP-1)、肿瘤坏死因子-α(TNF-α)、Fractalkine]、白细胞相关指标(WBC、WLCR、WSCR)、红细胞相关指标(RBC、HGB、HCT)、生化指标[总蛋白(TP)、白蛋白(ALB)、白球比(A/G)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、肌酐(CREA)、血尿素氮(BUN)、尿酸(UA)、钙(Ca)、磷(P)、钾(K)、钠(Na)、氯(Cl)]的水平,分析不同的饲育环境对动物福利的影响。结果：1.与对照组比较,腹腔注射操作造成Wistar大鼠CORT、IFN-γ、IFN-γ/IL-4、WSCR和肝脏hsp72mRNA表达量的升高,β-EP、CRP、IL-2、WBC和WLCR的降低；肌肉注射造成Wistar大鼠CORT、FN-γ、IFN-γ/IL-4、WSCR、RBC、HGB和hsp72mRNA表达量的升高,β-EP、CRP、IL-2、WBC和WLCR的降低；灌胃操作造成Wistar大鼠CORT、IFN-γ、IFN-γ/IL-4和hsp72mRNA表达量的升高,β-EP、CRP、IL-2、WBC、WLCR和HCT的降低,且差异均有显著性(P<0.01或P<0.05)。三种给药操作之间的比较显示,腹腔注射组的CORT、IL-2和hsp72mRNA表达量高于肌肉注射组；腹腔注射组的IFN-γ/IL-4低于灌胃组；肌肉注射组的RBC、HGB和HCT高于灌胃组,其IL-2低于灌胃组,且差异均有显著性(P<0.01或P<0.05)。2.与对照组比较,剪尾采血组的CORT、IFN-γ、IFN-γ/IL-4.WLCR和hsp72mRNA表达量升高,(3-EP、CRP、IL-2、WSCR、HGB和HCT降低；尾静脉切割采血组的CORT、IFN-γ、IFN-γ/IL-4和hsp72mRNA表达量升高,β-EP、WBCWSCR、HGB和HCT降低,且差异均有显著性(P<0.01或P<0.05)。两个采血组之间比较显示,剪尾采血组的IFN-γ/IL-4、WLCR和hsp72mRNA相对表达量高于尾静脉切割采血组；其CRP、IL-4和WSCR低于尾静脉切割采血组,且差异均有显著性(P<0.01或P<0.05)。3.与对照组比较,硬固定组的CORT、CRP、IL-2、IFN-γ/IL-4、RBC、HGB、HCT和hsp72mRNA表达量升高,β-EP、IL-4和WBC降低；软固定组的CRP、IL-2、IFN-γ/IL-4、RBC、HGB、HCT和hsp72mRNA表达量升高,β-EP、IL-4和WBC降低,且差异均有显著性(P<0.01或P<0.05)。两固定组之间比较显示,硬固定组的CORT、CRP IFN-γ、IFN-γ/IL-4、RBC、HGB、HCT和hsp72mRNA表达量均高于软固定组,其WBC水平低于软固定组,且差异均有显著性(P<0.01或P<0.05)。4.屏障环境中采用IVC饲育组(简称屏障环境+IVC组)与屏障环境中采用普通开放饲养笼饲育组(简称屏障环境组)比较,屏障环境+IVC组的IL-1β、Fractalkine、BUN、Ca和Ca/P高于屏障环境组,其CORT、IL-1α、IL-2、TNF-α、LIX、WBC和CREA(?)低于屏障环境组,且差异均有显著性(P<0.01或P<0.05)。普通环境中采用IVC饲育组(简称普通环境+IVC组)与屏障环境组比较,普通环境+IVC组的IL-lα、Fractalkine、RBC、HGB、HCT、TP、ALB、UA、Ca、Ca/P、K和CO2高于屏障环境组,其CORT、MCP-1、TNF-α、LIX、GLU和CREA低于屏障环境组,且差异均有显著性(P<0.01或P<0.05)。屏障环境+IVC组与普通环境+IVC组比较,屏障环境+IVC组的IL-1β、IL-10、MCP-1、RBC、HGB、HCT和BUN高于普通环境+IVC组,屏障环境+IVC组的IL-1a、WBC、ALB、ALT、UA和K低于普通环境+IVC组,且差异均有显著性(P<0.01或P<0.05)。结论：本研究表明,各种常规实验操作和不同的饲育环境对Wistar大鼠神经内分泌、免疫、血液生理等指标均有不同程度的影响。其中各种常规实验操作均使Wistar大鼠神经内分泌功能出现紊乱,免疫功能不同程度受抑,增加了动物对相关疾病的易感性,采血和固定操作还会破坏大鼠红细胞的生理平衡。可见,各种常规实验操作均会对Wistar大鼠的福利产生不利影响。比较同类实验操作对动物福利的影响程度发现,尾静脉切割采血优于剪尾采血,软固定优于硬固定。三种不同的饲育环境对Wistar大鼠福利的影响程度也不一致。采用屏障环境开放饲养笼饲育,大鼠容易受到各种室内环境因素的干扰,使动物健康受到潜在威胁。普通环境中采用IVC饲育,容易造成动物的缺氧,并会使动物的红细胞相关指标和肝、肾等器官的代谢功能出现异常。屏障环境中采用IVC饲育,对大鼠肾脏的代谢指标有一定影响。综合比较这三种饲育环境,屏障环境中采用IVC饲育为最佳动物饲育方式,屏障环境中采用开放饲养笼次之,普通环境中采用IVC对动物福利的损害最大,不推荐使用。此外,各种常规实验操作和饲育环境的不同所造成的Wistar大鼠各系统指标的变化,均可能会对相关实验研究产生一定的背景性干扰。本研究中采用的神经内分泌、免疫、血液生理生化指标、应激蛋白和应激分子能够反映动物的整体福利状况,可作为实验动物福利水平评价指标体系的一部分。更多还原

【Abstract】 Objective:There is a close relationship between environmental factors and laboratory animal welfare. Routine experimental procedures and rearing environment are two of the more important environmental factors that may affect laboratory animal welfare. Evaluating the effects of these environmental factors on laboratory animal will help to improve animal welfare. However, at present systemic research on these aspects are very rare. The research observed the influences of different routine experimental procedures and rearing environment on the physiological homeostasis of Wistar rats, and preliminarily analyzed these environmental factors on the welfare of Wistar rats and the background-interference which may be induced to related research.Methods:1. Wistar rats were divided into three groups by the type of dosing procedures, then the procedures of Intraperitoneal injection (group Inl), intramuscular injections (group In2) and intragastric injection (group In3) were separately executed to each group of the animals. After continuously handled for seven days, the neuroendocrine parameters(CORT, (3-EP), immune parameters (IL-2, IL-4, IFN-y, IFN-γ/IL-4),white blood cell index (WBC, WLCR, WSCR), red blood cell index (RBC, HGB, HCT),stress protein (CRP) and stress molecules(hsp72 mRNA expression) were measured to analyze the effects of these procedures on the welfare of Wistar rats.2. Wistar rats were divided into two groups by the type of blood sampling, then the procedures of blood sampling by tail tip cutting(group Bsl) and blood sampling by tail vein incision(group Bs2) were separately executed to each group of the animals. After continuously handled for seven days, the neuroendocrine parameters, immune parameters, white blood cell index, red blood cell index, stress protein and stress molecules were measured to analyze the effects of these procedures on the welfare of Wistar rats. 3. Wistar rats were divided into two groups by the type of restraint, then the procedures of hard restraint (group Rel) and soft restraint (group Re2) were separately executed to each group of the animals. After continuously handled for seven days, the neuroendocrine parameters, immune parameters, white blood cell index, red blood cell index, stress protein and stress molecules were measured to analyze the effects of these procedures on the welfare of Wistar rats.4. Wistar rats were divided into three groups by the type of rearing environment. Then the animals were separately raised by ordinary breeding cage in barrier environment (group P), individually ventilated cage (IVC) in barrier environment (group PI) and IVC in conventional environment (group TI). After raised for forty-five days, the neuroendocrine parameters(CORT,β-EP), immune parameters(IL-la, IL-1(3, IL-2, IL-4, IL-10, IL-13, IFN-γ, IFN-γ/IL-4, MCP-1, TNF-a, Fractalkine, LIX),white blood cell index(WBC, WLCR, WSCR),red blood cell index(RBC, HGB, HCT) and biochemical parameters (TP, ALB, A/G, ALT, AST, CREA, BUN, UA, Ca, P, K, Na, Cl)were measured to analyze the effects of different rearing environment on the welfare of Wistar rats.Results:1. Compared with control group, the level of CORT, IFN-γ, IFN-γ/IL-4, WSCR, hsp72 mRNA expression significantly increased and the level of P-EP, CRP, IL-2, WBC, WLCR significantly decreased in group Inl. Compared with control group, the level of CORT, IFN-γ, IFN-γ/IL-4, WSCR, RBC, HGB, hsp72 mRNA expression significantly increased and the level ofβ-EP, CRP, IL-2, WBC, WLCR significantly decreased in group In2. Compared with control group, the level of CORT, IFN-y, IFN-γ/IL-4, hsp72 mRNA expression significantly increased and the level ofβ-EP, CRP, IL-2, WBC, WLCR, HCT significantly decreased in group In3. Compared with group In2, group Inl showed higher levels of CORT, IL-2 and hsp72 mRNA expression. Compared with group In3, group In1 showed lower levels of IFN-y/IL-4. Compared with group In3, group In2 showed higher levels of RBC, HGB and HCT, lower levels of IL-2. All the differences in this research were significant.2. Compared with control group, the level of CORT, IFN-y, IFN-y/IL-4, WLCR, hsp72 mRNA expression significantly increased and the level ofβ-EP, CRP, IL-2, WSCR, HGB, HCT significantly decreased in group Bsl. Compared with control group, the level of CORT, IFN-y, IFN-γ/IL-4 and hsp72 mRNA expression expression significantly increased and the level ofβ-EP, WBC, WSCR, HGB, HCT significantly decreased in group Bs2. Compared with group Bs2, group Bsl showed higher levels of IFN-y/IL-4, WLCR and hsp72 mRNA expression, lower levels of CRP, IL-4 and WSCR. All the differences in this research were significant.3. Compared with control group, the level of CORT, CRP, IL-2, IFN-y/IL-4, RBC, HGB, HCT, hsp72 mRNA expression significantly increased and the level ofβ-EP, IL-4 and WBC significantly decreased in group Rel. Compared with control group, the level of CRP, IL-2, IFN-y/IL-4, RBC, HGB, HCT and hsp72 mRNA expression significantly increased and the level ofβ-EP, IL-4 and WBC significantly decreased in group Re2. Compared with group Re2, group Rel showed higher levels of CORT, CRP IFN-y, IFN-γ/IL-4, RBC, HGB, HCT and hsp72 mRNA expression, lower levels of WBC. All the differences in this research were significant.4. Compared with group P, group PI showed higher levels of IL-1β, Fractalkine, BUN, Ca and Ca/P, lower levels of CORT, IL-la, IL-2, TNF-a, LIX, WBC and CREA. Compared with group P, group TI showed higher levels of IL-la, Fractalkine, RBC, HGB, HCT, TP, ALB, UA, Ca, Ca/P, K and CO2, lower levels of CORT, MCP-1, TNF-a, LIX, GLU and CREA. Compared with group TI, group PI showed higher levels of IL-1β, IL-10, MCP-1, RBC, HGB, HCT and BUN, lower levels of IL-la, WBC, ALB, ALB, UA and K. All the differences in this research were significant.Conclusion: The studies showed that the neuroendocrine, immune and blood physical indexes of Wistar rats were varyingly affected by these routine experimental procedures and different rearing environments. All the routine experimental procedures have induced neuroendocrine disorder and immune suppression in Wistar rats, and increased susceptibility to diseases in the animals. Furthermore, the red blood physiological homeostasis of the rats was destroyed by blood sampling and restraint procedures. These results indicate that routine experimental procedures could induce negative influence on the welfare of Wistar rats. Comparing the same type of the procedures on the effects of animal welfare, blood sampling by tail vein incision is better than blood sampling by tail tip cutting, soft restraint is better than hard restraint. The three different rearing environments also differently affected the welfare of Wistar rats. The rats reared in the breeding cage in barrier environment easily interfered by different kinds of external environmental factors that may make potential threats to their health. The rats reared in IVC in conventional environment prone to anoxia and may induce abnormal of the anabolic function of their liver, kidney and other organs. The rats reared in IVC in barrier environment made little influence on their metabolic function. Comparing the three different kinds of rearing environment, rearing animal in IVC in barrier environment is the best rearing method, rearing animal in ordinary breeding cage in barrier environment is the second choice, rearing animal in IVC in conventional environment is not recommended. Furthermore, these changes may induce background-interference on some experimental results. The neuroendocrine, immune, blood physiological and biochemical indexes, stress protein and stress molecules that involved in the experiments can be used to evaluate the welfare of laboratory animals.更多还原